Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy.

Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation ((3)H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80 µM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140 µM) markedly decreased MTT absorbance (40%) at 120 h. VR or VS treatment inhibited (3)H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP(+) puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.

Intestinal tract injury by drugs: Importance of metabolite delivery by yellow bile road.

Drug secretion into bile is typically considered a safe route of clearance. However, biliary delivery of some drugs or their reactive metabolites to the intestinal tract evokes adverse consequences due to direct toxic actions or indirect disruption of intestinal homeostasis. Biliary concentration of the chemotherapy agent 5-fluorodeoxyuridine (FUDR) and other compounds is associated with bile duct damage while enterohepatic cycling of antibiotics contributes to the disruptions of gut flora that produce diarrhea. The goal of this review is to describe key evidence that biliary delivery is an important factor in the intestinal injury caused by representative drugs. Emphasis will be given to 3 widely used drugs whose reactive metabolites are plausible causes of small intestinal injury, namely the nonsteroidal anti-inflammatory drug (NSAID) diclofenac, the immunosuppressant mycophenolic acid (MPA), and the chemotherapy agent irinotecan. Capsule endoscopy and other sensitive diagnostic techniques have documented a previously unappreciated, high prevalence of small intestinal injury among NSAID users. Clinical use of MPA and irinotecan is frequently associated such severe intestinal injury that dosage must be reduced. Observations from clinical and experimental studies have defined key events in the pathogenesis of these drugs, including roles for multidrug resistance-associated protein 2 (MRP2) and other transporters in biliary secretion and adduction of enterocyte proteins by reactive acyl glucuronide metabolites as a likely mechanism for intestinal injury. New strategies for minimizing the adverse intestinal consequences of irinotecan chemotherapy illustrate how basic information about key events in the biliary secretion of drugs and the nature of their proximate toxicants can lead to safer protocols for drugs.

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum.

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.

Proteomic characterization of metabolites, protein adducts, and biliary proteins in rats exposed to 1,1-dichloroethylene or diclofenac.

A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH(2)COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCE-treated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.